It has recently become possible to trap individual fluorescent biomolecules in aqueous solution by using real-time tracking and active feedback to suppress Brownian motion. We propose areas of investigation in which anti-Brownian electrokinetic (ABEL) trapping of single molecules is likely to lead to significant new insights into biomolecular dynamics.

methods

/pdf/the-cat-that-caught-the-canary-what-to-do-with-single-3kyi9osemi.pdf

Experimental study of memory erasure in a double-well potential

The cat that caught the canary: what to do with single-molecule trapping.

I present two applications of fluorescence spectroscopy in biophysics. The first is an instrument, the anti-Brownian electrokinetic (ABEL) trap, which is capable of trapping individual small molecules in aqueous solution at room temperature. The second is an investigation of the bending mechanics of doublestranded DNA using a novel DNA structure called a “molecular vise”. Both projects take advantage of the sensitivity and specificity of fluorescence spectroscopy, and both benefit from the interplay of experimental work with theoretical and computational modeling. The ABEL trap uses fluorescence microscopy to track a freely diffusing particle, and applies real-time electrokinetic feedback forces to oppose observed motion. Small molecules are difficult to trap because they diffuse quickly and because their fluorescence emission is typically weak. I describe the experimental and algorithmic approaches that enabled small-molecule fluorophores to be trapped at room temperature. I additionally derive and discuss the theory of the molecules’ behavior in the trap; this mathematical work informed the design of the trapping algorithm and additionally enabled trapped molecules to be distinguished on the basis of their diffusion coefficient and electrokinetic mobility. Molecular vises are DNA hairpins that use the free energy of hybridization to exert a compressive force on a sub-persistence length segment of double-stranded iv DNA. In response to the applied force, this “target strand” may either remain straight or bend, depending on its flexibility and length. Experimentally, the conformation can be monitored via Förster resonance energy transfer (FRET) between appended fluorophores. The experimental results quantitatively matched the predictions of the classic wormlike chain (WLC) model of DNA elasticity at lowto-moderate salt concentrations. Higher ionic strength induced an apparent softening of the DNA which was best accounted for by a high-curvature “kinked” state. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA and provides a platform for studying the effects of chemical modifications and small-molecule or protein binding on these properties.

background

Electrokinetic Trapping of Single Molecules, and Euler Buckling and Nonlinear Kinking of DNA

http://absimage.aps.org/image/MAR11/MWS_MAR11-2010-001934.pdf

Experimental study of memory erasure in a double-well potential

Citations

The cat that caught the canary: what to do with single-molecule trapping.

Electrokinetic Trapping of Single Molecules, and Euler Buckling and Nonlinear Kinking of DNA

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